The Bacillus subtilis tyrS gene is a member of a large group of aminoacyl-tRNA synthetase, amino acid biosynthesis and transporter genes, designated the T box family, that are regulated by a unique transcription termination control system. Expression of each gene is dependent on interaction of the leader region of the transcript with a specific uncharged tRNA. This interaction promotes formation of an antiterminator structure, preventing premature termination of transcription. We have identified over 250 transcriptional units, primarily from Gram-positive bacteria, with leader regions exhibiting the conserved elements characteristic of members of this family. Several important pathogens, including Bacillus anthracis, Staphylococcus, Streptococcus, Enterococcus and Mycobacterium, are represented in this group. Since most of the regulated genes encode essential proteins, this system represents a potential target for antibiotic development. Expression of each gene in the family is dependent on pairing of the anticodon of the inducer tRNA with a single codon, the "specifier sequence," in the leader, and on pairing of acceptor end of the tRNA with a bulged region of the antiterminator; these pairings are necessary but not sufficient for efficient antitermination. Phylogenetic analysis of the leaders has revealed a number of elements the structure of which can be predicted based on similarity to other RNAs. Novel variations on the arrangement of conserved leader elements has also been uncovered; some of these variations are suggestive of variability in the molecular mechanism of antitermination. The next project period will focus on using the phylogenetic data as a basis for efforts to uncover additional structural and mechanistic features of the system. The approaches used will include computational analyses of our extensive aligned leader RNA and tRNA database, and structural studies of leader elements in parallel to our successful analysis of the antiterminator domain. Genetic and biochemical approaches will be directed to the refinement of the required leader and tRNA elements, and for identification of possible additional factors.